Leptin和HO-1在脑缺血再灌注损伤中的相互作用及机制研究

Cerebral ischemia and reperfusion injury (CIRI) is mainly caused by neuronal injury or necrosis. Accumulating evidence suggests that astrocytes, leptin and heme oxygenase-1 (HO-1) may protect neurons against brain ischemic injury. However, Recent evidence suggests over-activated astrocytes and over-expressed HO-1 have the harmful effects in cerebral ischemic stroke. Our preliminary experiments show that exogenous leptin could significantly improve the nerve function, reduced the infart volume after CIRI and that leptin could significantly decrease the HO-1 level. This phenomenon has caused us great concern. Does leptin have a reverse regulation effect towards HO-1 during CIRI? In turn, how about the leptin expression after exogenous administration of HO-1? Till now, there is no literature about the interrelation between leptin and HO-1. So, this project aims to clarify whether leptin and HO-1 are interrelated in terms of time and space during CIRI. Herein, we intend to investigate the changes of leptin and HO-1 expression and the interrelation between them after acute cerebral ischemia/reperfusion and hypoxia/reoxygenation injury, using leptin and HO-1 gene-deficient mice and astrocytes, from levels of the animals, tissues and cells, Furthermore, we will test whether the interrelation is regulated by MAPK/ERK or Nrf2. This project may provide a theoretical basis for the study of cerebral infarction lesion repair mechanism.

脑缺血再灌注损伤多由神经元损伤或坏死所致，星形胶质细胞（As）、瘦素（Leptin）和血红素加氧酶-1（HO-1）均具有神经保护功能，不同活化程度的As，不同表达水平的HO-1，对神经元或发挥保护作用，或产生损伤效应。前期实验表明，急性缺血缺氧损伤后，外源性Leptin，有利于受损神经元功能恢复和脑梗塞病灶好转，同时HO-1表达显著降低。这一现象引起我们高度关注。难道Leptin对HO-1具有反向调节作用？反过来，外源性HO-1对Leptin的表达有何影响呢？目前文献中关于二者相互关系尚无报道。为明确Leptin与HO-1在时间和空间上是否相互关联，本课题拟采用Leptin与HO-1基因缺陷小鼠及As为研究对象，从整体、组织和细胞三个水平，探讨急性损伤后，Leptin和HO-1的表达变化及其相互关系，并验证其是否受MAPK/ERK或Nrf2调控，从而为研究脑梗塞病灶的修复机制提供理论依据。

脑缺血再灌注损伤（CIRI）多由神经元损伤或坏死所致。Leptin和HO-1对受损神经元具有保护作用。目前关于二者相互关系尚无报道。本课题采用Leptin与HO-1基因缺陷小鼠及胶质细胞为研究对象，从整体、组织和细胞三个水平，探讨了急性CIRI后Leptin和HO-1表达变化及其相互关系，这对于认识Leptin和HO-1如何参与神经元损伤的保护、胶质细胞如何挽救邻近神经元免受局部缺血所致的死亡以及脑梗塞病灶的修复机制具有重要意义。取得成果如下：.1. Leptin对HO-1的调控.（1）制备了小鼠局灶性CIRI模型，外源性给与Leptin能够显著降低小鼠死亡率、减小脑梗死体积、改善神经功能评分；（2）Leptin发挥CIRI保护作用的同时能够促进缺血半影区HO-1表达；（3）无论WT还是ob小鼠，海马神经元几乎没有HO-1表达，MC细胞有较高HO-1表达，且ob鼠MC细胞中HO-1表达显著低于WT；（4）BV-2细胞缺氧复氧（H/R）损伤后，外源性给与Leptin能显著提高HO-1表达并呈一定剂量依赖性；（5）外源性给与Leptin能够显著增强BV-2小胶质细胞在损伤状态下的吞噬能力及膜边缘波动效应。.2. HO-1对Leptin的调控..（1）成功构建了HO-1高低表达基因缺陷小鼠，发现WT与HO-1基因缺陷小鼠海马神经元结构无明显差异；（2）考察了三种小鼠脑、心脏和脂肪组织HO-1、Leptin表达情况，发现脑中表达最高，HO-1对Leptin表达具有一定调控作用。（3）发现WT小鼠皮层和海马神经元本身HO-1表达较低，ZnPP/CoPP能够显著降低/升高HO-1水平，HO-1表达降低/升高时，Leptin表达也随着降低/升高;（4）发现H/R损伤后不同时间点HO-1高表达小鼠脑片的Leptin水平呈升高趋势，而HO-1低表达小鼠的Leptin水平一直在下降；（5）免疫荧光发现H/R损伤后U87细胞HO-1表达升高，Leptin和OB-R表达降低，ZnPP/CoPP能够显著降低/升高HO-1和Leptin表达，OB-R表达呈相反趋势，细胞上清Leptin和OB-R表达变化与免疫荧光一致。.3. 结论：急性CIRI后，Leptin和HO-1存在正向调节关系，外源性给与Leptin/HO-1能够促进HO-1/Leptin表达，二者协同发挥神经保护作用。